FEBS 22469 FEBS Letters 457 (1999) 291^297 CORE Metadata, citation and similar papers at core.ac.uk

Provided by Elsevier - Publisher Connector Invited minireview Novel reactions involved in energy conservation by methanogenic archaea

Uwe Deppenmeier*, Tanja Lienard, Gerhard Gottschalk

Institut fu«r Mikrobiologie und Genetik, Georg-August-Universita«t, Grisebachstr. 8, 37077 Go«ttingen, Germany

Received 12 July 1999

cales, Methanomicrobiales and Methanopyrales which use Abstract Methanogenic archaea of the order Methanosarci- nales which utilize C compounds such as methanol, methyl- only H2+CO2 or formate as substrates (hydrogenotrophic 1 methanogens). In contrast, members of the order Methano- amines or H2+CO2, employ two novel membrane-bound electron transport systems generating an electrochemical proton sarcinales are more versatile and utilize methanol, methyl- gradient: the H2 :heterodisulfide oxidoreductase and the amines, methylthiols or acetate (methylotrophic methano- F420H2 :heterodisulfide oxidoreductase. The systems are com- gens); some of them also grow on H2+CO2 [4]. posed of the heterodisulfide reductase and either a membrane- bound hydrogenase or a F420H2 dehydrogenase which is 2. Methanophenazine ^ a new cofactor of methanogenesis functionally homologous to the proton-translocating NADH dehydrogenase. Cytochromes and the novel electron carrier Several unusual cofactors are involved in methanogenesis; methanophenazine are also involved. In addition, the methyl- their structures were elucidated during the last decade [2,5,6]. H4MPT:HS-CoM methyltransferase is bioenergetically rele- vant. The enzyme couples methyl group transfer with the Methanofuran (MF), tetrahydromethanopterin (H4MPT) and translocation of sodium ions and seems to be present in all coenzyme M (HS-CoM, 2-mercaptoethanesulfonate) are C1 methanogens. The proton-translocating systems with the par- carriers. Coenzyme B (HS-CoB, 7-mercaptoheptanoylthreo- ticipation of cytochromes and methanophenazine have been nine phosphate) functions as electron donor in the terminal found so far only in the Methanosarcinales. reaction of methanogenesis. Coenzyme F420 is a 5-deaza£avin z 1999 Federation of European Biochemical Societies. derivative (E0P = 3360 mV) and is the central electron carrier in the cytoplasm of methanogens. Very recently, a new redox- Key words: Methanogenesis; Methanophenazine; active component was isolated from the cytoplasmic mem- Proton translocation; Sodium ion translocation; brane of Methanosarcina (Ms.) mazei Go«1; it is referred to Hydrogenase; F H dehydrogenase 420 2 as methanophenazine (Fig. 1). This cofactor represents a 2- hydroxyphenazine derivative which is linked via an ether bridge to a pentaisoprenoid side chain [7]. Methanophenazine 1. Introduction is the ¢rst example of a phenazine chromophore produced by archaea. Moreover, it is the only phenazine derivative that Methanogenic organisms belong to the kingdom of archaea participates in respiratory chains of living cells (see Sections and are widespread in anoxic environments. They have re- 4.1 and 4.2). ceived much attention due to their ecological and biochemical features. (1) The process of methanogenesis is important for 3. Pathways of methanogenesis the global carbon cycle since it represents the terminal step in the anaerobic breakdown of organic matter under sulfate-lim- The pathways of methanogenesis from the above-men- iting conditions. (2) Large amounts of CH4 escape into the tioned substrates are summarized in Fig. 2. Methanogenesis atmosphere where it acts as a greenhouse gas [1]. (3) The from H2+CO2 proceeds according to the following equation pathway of methanogenesis comprises unusual enzymes and [2,8]: cofactors [2]. (4) The mechanisms of energy conservation in- 0 volve novel ion-translocating reactions [3]. CO2 ‡ 4H2 ! CH4 ‡ 2H2O vG0 ˆ 3130 kJ=mol† 1† On the basis of the substrates utilized, methanogens can be divided into two major groups. The ¢rst group comprises the The pathway starts with the H2- and MF-dependent reduction organisms of the orders Methanobacteriales, Methanococ- of CO2 to formyl-MF. The endergonic reaction is catalyzed by a formyl-MF dehydrogenase and is driven by an electro- chemical ion gradient (see Section 4.3). The formyl group is *Corresponding author. Fax: (49) (551) 393808. then transferred to H MPT and the resulting formyl-H MPT E-mail: [email protected] 4 4 is stepwise reduced to methyl-H4MPT. The electrons are de- Abbreviations: CoB-SH, 7-mercaptoheptanoylthreonine phosphate; rived from reduced F420 (F420H2) which is produced by the CoM-SH, 2-mercaptoethanesulfonate; F420,(N-L-lactyl-Q-L-glutam- F420-reducing hydrogenase. The methyl group of methyl- yl)-L-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5- H MPT is then transferred to HS-CoM by the methyl- deazaribo£avin-5P-phosphate; F H , reduced F ; Mc., Methano- 4 420 2 420 H MPT:HS-CoM methyltransferase. The exergonic reaction coccus; Mb., Methanobacterium; Ms., Methanosarcina; MF, metha- 4 (vG0P = 329 kJ/mol) is coupled to the formation of an electro- nofuran; H4MPT, tetrahydromethanopterin; vWH‡ , electrochemical proton gradient; vWNa‡ , electrochemical sodium ion gradient chemical sodium ion gradient (vWNa‡, see Section 6). The ¢nal

0014-5793 / 99 / $20.00 ß 1999 Federation of European Biochemical Societies. All rights reserved. PII: S0014-5793(99)01026-1

FEBS 22469 30-8-99 292 U. Deppenmeier et al./FEBS Letters 457 (1999) 291^297

4.1. H2 :heterodisul¢de oxidoreductase When methanogens are grown on H2+CO2 molecular hy- drogen is used for the reduction of CoM-S-S-CoB (Fig. 2). In Fig. 1. Structure of methanophenazine. Methanosarcina strains this reduction is catalyzed by the membrane-bound H2 :heterodisul¢de oxidoreductase system [3,9]. The oxidation of H2 by the F420-non-reducing hydro- genase is the initial reaction (Fig. 3). This enzyme was isolated step in methanogenesis is the reduction of CH3-S-CoM to from Ms. barkeri [10] and Ms. mazei Go«1 [3]. The puri¢ed CH4 which can be divided into two partial reactions. First proteins consist of two di¡erent subunits with molecular methyl-S-CoM is reductively cleaved by the methyl-S-CoM masses of approximately 60 and 40 kDa and contain a re- reductase using HS-CoB as reductant. The reaction results dox-active Ni ion and FeS clusters but no £avins. Ms. mazei in the formation of methane and a heterodisul¢de (CoM-S- Go«1 contains two sets of genes (the vho and the vht operons) S-CoB) from HS-CoM and HS-CoB. In a second reaction which encode F420-non-reducing hydrogenases ([11], see also CoM-S-S-CoB is reduced by the heterodisul¢de reductase. Section 4.3). The vhoGAC-encoded enzyme is believed to be The reducing equivalents are derived from H2 and channeled part of the H2 :heterodisul¢de oxidoreductase [3]. The de- to the heterodisul¢de reductase by a membrane-bound elec- duced amino acid sequences revealed that the small (VhoG) tron transport system (see Section 4.1). and the large subunit (VhoA) of the protein are homologous When cells grow on methanol, methylamines or methyl- to corresponding polypeptides of membrane-bound NiFe hy- thiols, the substrates undergo disproportionation to CH4 drogenases from several bacteria [12]. A third gene (vhoC) and CO2 [3], e.g. methanol is converted according to the fol- belonging to the hydrogenase operon from strain Go«1 enco- lowing equation: des a b-type cytochrome (cyt b1) which probably functions as primary electron acceptor of the core enzyme consisting of 0 4CH3OH ! 3CH4 ‡ 1CO2 ‡ 2H2O vG0 ˆ 3106 kJ=mol† VhoG and VhoA [13]. The heterodisul¢de reductase is the 2† second component of the H2 :heterodisul¢de oxidoreductase and reduces CoB-S-S-CoM. The enzyme from Methanosarcina In the oxidative branch of the pathway (Fig. 2) one out of four methyl groups is oxidized to CO2 by the reversed CO2 reduction route. The series of reactions starts with the forma- tion of CH3-S-CoM and methyl group transfer to H4MPT as catalyzed by the methyl-H4MPT:HS-CoM-methyltransferase. The endergonic reaction (vG0P = 29 kJ/mol) is driven by an electrochemical sodium ion gradient (see Section 6). The for- mation of methyl-H4MPT is followed by the stepwise oxida- tion to formyl-H4MPT. Reducing equivalents derived from these reactions are used for F420 reduction. After transfer of the formyl group to MF the formyl-MF dehydrogenase cata- lyzes the oxidation of CHO-MF to CO2 and MF (see Section 4.3). In the reductive branch of the pathway three out of four methyl groups are transferred to HS-CoM by methanol-spe- ci¢c methyltransferases. Again, the HS-CoB-dependent reduc- tion of methyl-S-CoM leads to the formation of CH4 and CoM-S-S-CoB. The latter component, however, is reduced by an electron transport system which does not occur in hydrogenotrophic methanogens, the membrane-bound F420H2 :heterodisul¢de oxidoreductase (see Section 4.2). Methylamines and methylthiols are converted in the same manner (Fig. 2) with the exception that methyl transfer to HS-CoM is catalyzed by substrate-speci¢c methyltransferases [8]. Methanogenesis from acetate is not reviewed here. Su¤ce it to say that the methyl group of acetate enters the methano- genic pathway at the level of methyl-H4MPT and that reduc- ing equivalents for heterodisul¢de reduction are provided by oxidation of CO to CO2 (for review see [8,9]).

4. Redox-driven ion translocation in methylotrophic Fig. 2. Pathways of methanogenesis from C1 compounds. Black ar- methanogens and function of methanophenazine rows, methane formation from H2+CO2 ; open arrows, methanogen- esis from methylated substrates. Reactions catalyzed by energy Transmembrane electrochemical ion gradients used for transducing enzymes are boxed. MF, methanofuran; H4MPT, tetra- hydromethanopterin; F , oxidized form of coenzyme F ;F H , ATP synthesis in methanogenic archaea are generated by 420 420 420 2 reduced F420 ; HS-CoM, 2-mercaptoethanesulfonate; HS-CoB, 7- three di¡erent electron transport systems which will be de- mercaptoheptanoylthreonine phosphate; CoB-S-S-CoM, heterodisul- scribed below. ¢de of HS-CoM and HS-CoB.

FEBS 22469 30-8-99 U. Deppenmeier et al./FEBS Letters 457 (1999) 291^297 293 species is composed of two subunits (HdrDE) [9,14]. HdrE represents a membrane-integral b-type cytochrome (cyt b2) and contains two distinct heme groups. HdrD is the catalytic subunit of the heterodisul¢de reductase and contains two Fe4S4 clusters. After the elucidation of the composition and properties of the key components of the H2 :heterodisul¢de oxidoreductase the most interesting question concerned the nature of the elec- tron carrier mediating electron transfer from the F420-non-re- ducing hydrogenase to the heterodisul¢de reductase. This problem was solved with the discovery of methanophenazine (see Section 2). The component is very hydrophobic and can- not be employed for in vitro assays in aqueous bu¡er systems. Fortunately, the water-soluble 2-OH-phenazine can be used instead. It is reduced by molecular hydrogen as catalyzed by the F420-non-reducing hydrogenase. Furthermore, the mem- brane-bound heterodisul¢de reductase uses dihydro-2-OH- phenazine as electron donor for the reduction of CoB-S-S- CoM [7]. Thus, the process of electron transfer from molec- ular hydrogen to the heterodisul¢de can be divided into two partial reactions:

H2 ‡ 2-OH-phenazine ! dihydro-2-OH-phenazine 3† dihydro-2-OH-phenazine ‡ CoM-S-S-CoB !

HS-CoM ‡ HS-CoB ‡ 2-OH-phenazine 4†

Both exergonic reactions (vG0Peq: 3 = 331.8 kJ/mol, vG0Peq: 4 = 310.6 kJ/mol) are coupled by washed inverted vesicles of Ms. mazei Go«1 with the transfer of protons across Fig. 3. Tentative scheme of the proton-translocating the cytoplasmic membrane [15]. The maximal H‡/2e3 ratio H2 :heterodisul¢de oxidoreductase and the F420H2 :heterodisul¢de oxidoreductase of Methanosarcina strains. MPhen, methanophena- was 0.9 for each reaction. Thus, the H :heterodisul¢de oxi- 2 zine; MPhenH2, dihydromethanophenazine; VhoG, 40-kDa subunit doreductase system comprises two di¡erent proton-translocat- of the F420-non-reducing hydrogenase; VhoA, 60-kDa subunit of ing segments. The ¢rst one involves the 2-OH-phenazine-de- the F420-non-reducing hydrogenase; VhoC, cytochrome b1 encoded pendent hydrogenase and the second one the heterodisul¢de by the third gene (vhoC) of the hydrogenase operon; [Fe-Ni], bimet- allic Fe-Ni cluster; [FeS], iron-sulfur cluster; HdrDE, subunits of reductase. the heterodisul¢de. The question marks in the membrane-integral The H‡/2e3 stoichiometries of reactions 3 and 4 add up to part of the F420H2 dehydrogenase indicate that the mechanisms of 1.8 which corresponds to the e¤ciency of the overall electron electron transfer and H‡ translocation are unknown. transport from H2 to CoM-S-S-CoB as determined earlier [3]. However, 3^4 H‡/2e3 were transferred by whole cell prepa- rations of Ms. barkeri during methane formation from meth- the protein surface where the reduction of the electron accept- anol+H2 [16]. This discrepancy is explained by the fact that or takes place. His and Glu residues which are arranged be- about 50% of the membrane structures present in the vesicle tween the active center and the surface of the protein are preparations of Ms. mazei Go«1 catalyze electron transport not involved in the release of protons into the periplasm in the coupled to the generation of a proton gradient [15]. course of the reaction cycle. These amino acids are highly The course of electron transport and the sites of H‡ trans- conserved among NiFe hydrogenases and are also present in location of the H2 :heterodisul¢de oxidoreductase system are the F420-non-reducing hydrogenase from Ms. mazei indicating summarized in Fig. 3. As already mentioned the F420-non- that the mechanism of proton release may be identical in this reducing hydrogenase from Ms. mazei Go«1 is highly homol- methanogenic organism. ogous to corresponding enzymes of several bacteria. Thus, Taking together these ¢ndings the mechanism of redox- ‡ some features of the bacterial proteins might also be impor- driven H translocation as catalyzed by the F420-non-reducing tant for the hydrogenase from strain Go«1. The mechanism of hydrogenase from Ms. mazei Go«1 can be described as follows ‡ H release and electron transfer became evident from several (Fig. 3): H2 is oxidized by VhoA which contains the bimet- biophysical and genetic studies [17] as well as from the anal- allic Ni-Fe center. Two protons are transferred via histidine ysis of the three-dimensional structure of the periplasmic hy- and glutamate residues to the surface of VhoA and are re- drogenase from Desulfovibrio gigas [18]. According to these leased in the periplasm. The electrons are transferred to sub- studies, the active center located in the large subunit contains unit VhoG and channeled via FeS clusters to VhoC. From a bimetallic Ni-Fe cluster which catalyzes the heterolytic inhibitor studies with diphenyleneiodonium chloride it is cleavage of molecular hydrogen. Three FeS clusters present known that the heme b-containing subunit and methanophen- in the small subunit are distributed along a straight line and azine interact directly [13]. Thus, it is reasonable to assume provide an electron channel going from the active center to that the active center for methanophenazine reduction is lo-

FEBS 22469 30-8-99 294 U. Deppenmeier et al./FEBS Letters 457 (1999) 291^297

5 Fig. 4. Hypothetical scheme of the organization of the membrane-bound N -methyl-H4MPT:CoM methyltransferase. Data on the localization of MtrH were taken from [49] and of MtrA from [47]. The localization of all subunits was con¢rmed by mini cell expression experiments in E. coli [45]. cated on VhoC. The protons necessary for the reduction of erated in methylotrophic methanogens during methanogenesis methanophenazine may be derived from the cytoplasm so that from methanol and methylamines (see Section 3). The this segment of the electron transport chain results in the F420H2 :heterodisul¢de oxidoreductase, which is composed of generation of two scalar protons. aF420H2 dehydrogenase and the heterodisul¢de reductase, The reduction of CoB-S-S-CoM is catalyzed by the hetero- reoxidizes F420H2 at the expense of CoM-S-S-CoB reduction disul¢de reductase (Fig. 3). The reaction resembles the process (Fig. 3). The overall reaction has been shown to be competent of sulfur/polysul¢de respiration as performed by several bac- in driving proton translocation across the cytoplasmic mem- teria and archaea. It is proposed that the reaction mechanism brane [3]. Similar to the H2 :heterodisul¢de oxidoreductase, of the heterodisul¢de reductase is similar to that of the ferre- electron transport and H‡ translocation are strictly coupled, doxin-thioredoxin reductase from plants and cyanobacteria as indicated by stoichiometries of 4 H‡/2e3. Recent studies [9]. In contrast to many other £avin-containing disul¢de re- revealed that 2-OH-phenazine is reduced by the membrane- ductases, the active centers of the above-mentioned enzymes bound F420H2 dehydrogenase from Ms. mazei Go«1 with only possess Fe4S4 clusters and a redox-active disul¢de. The F420H2 as electron donor. The resulting dihydro-2-OH-phena- ferredoxin-thioredoxin reductase catalyzed the reduction of zine acts again as substrate for the heterodisul¢de reductase thioredoxin with reduced ferredoxin by two one-electron (see Section 4.1) indicating that the F420H2-dependent hetero- transfer reactions indicating a one-electron-reduced intermedi- disul¢de reduction consists of two partial reactions [7]: ate in the reaction cycle [19]. F H ‡ 2-OH-phenazine ! As depicted in Fig. 3 it is assumed that dihydromethano- 420 2 phenazine binds to the membrane-integral subunit HdrE of the heterodisul¢de reductase where the reduced electron car- F420 ‡ dihydro-2-OH-phenazine 5† rier is stepwise oxidized. Electrons are transferred to the heme groups of the polypeptide and two protons are released at the dihydro-2-OH-phenazine ‡ CoM-S-S-CoB ! outer aspect of the cytoplasmic membrane. In a second step the electrons are channeled to FeS-clusters of the membrane- HS-CoM ‡ HS-CoB ‡ 2-OH-phenazine 4† associated subunit HdrD and CoM-S-S-CoB is reduced to HS-CoM and HS-CoB. Protons necessary for this reaction As in the H2-dependent system both reactions contribute to are provided by the cytoplasm. energy conservation with H‡/2e3 ratios of 1.8 each (unpub- In summary, both the reduction of methanophenazine as lished results). The sum of these values is in the same range as ‡ 3 catalyzed by the F420-non-reducing hydrogenase and the oxi- the ratio of 4 H /2e determined earlier for the whole system dation of dihydromethanophenazine as catalyzed by the het- [3]. erodisul¢de reductase contribute to the generation of an elec- All key enzymes of this methanogenic electron transport trochemical proton gradient (vWH‡ ) which can be taken chain have been isolated and characterized. The F420H2 dehy- advantage of by the A1A0-type ATP synthase [20]. drogenase from Ms. mazei Go«1 with a molecular mass of 115 kDa contains iron-sulfur clusters and FAD [3]. The enzyme is 4.2. F420H2 :heterodisul¢de oxidoreductase very similar to the corresponding protein from Methanolobus As already mentioned F420H2 and CoM-S-S-CoB are gen- tindarius [21]; the solubilized complex is composed of ¢ve

FEBS 22469 30-8-99 U. Deppenmeier et al./FEBS Letters 457 (1999) 291^297 295 di¡erent subunits with molecular masses of 40, 37, 22, 20 and Ms. mazei Go«1 is connected to the formyl-MF dehydrogenase 16 kDa. A F420H2 dehydrogenase has also been isolated from [3]. According to these ¢ndings, the formation of formyl-MF the sulfate-reducing archaeon Archaeoglobus fulgidus [22]. Re- could proceed as follows: (1) H2 is oxidized by the vht-en- cently, the F420H2 :heterodisul¢de oxidoreductase could be re- coded hydrogenase. The electrons are transferred to the heme constituted by the combination of puri¢ed F420H2 dehydro- b-containing subunit VhtC. (2) The polyferredoxin FmdF genase from Ms. mazei Go«1, 2-OH-phenazine and puri¢ed could function as mediator of electron transfer from VhtC heterodisul¢de reductase from Ms. thermophila [23]. to the formyl-MF dehydrogenase. The latter protein then re- The F420H2 dehydrogenase is functionally homologous to duces enzyme-bound carboxy-MF to formyl-MF. The redox the NADH:ubiquinone oxidoreductase. Both enzymes reveal potential di¡erence between cytochrome b and FmdF may be a complex subunit composition and contain FeS clusters and overcome by an unknown mode of reversed electron transport £avins [24]. F420H2 and NADH are reversible hydride donors driven by vWH‡ or vWNa‡ . The formyl-MF dehydrogenase with comparable mid-point potentials. Electrons derived from system also catalyzes the reverse reaction during methanogen- the oxidation process are transferred to quinones and meth- esis from methanol or methylamines, where it is involved in anophenazine, respectively, which are similar in possessing the dehydrogenation of formyl-MF to CO2 and MF (Fig. 2). isoprenoid side chains that enable them to move in the hydro- In this pathway the enzyme functions as a primary ion pump, carbon phase of the cytoplasmic membrane. It has been thereby energizing the cytoplasmic membrane of the organ- shown that mitochondrial and bacterial NADH:ubiquinone isms. It is still unknown how electrons derived from this re- oxidoreductases as well as the F420H2 dehydrogenase are in- action are channeled to the heterodisul¢de reductase. hibited by diphenyleneiodonium chloride [13,25]. The se- quencing of the entire genome of Ms. mazei Go«1 is currently 5. Redox-driven energy conservation in hydrogenotrophic in progress (Genomics Laboratory Go«ttingen). First results methanogens indeed unravel a close relationship of NADH dehydrogenase and F420H2 dehydrogenase. The study of the enzymes is es- In contrast to methylotrophic methanogens, information on pecially interesting in the view of their proton-translocating the mechanism of energy conservation in obligate hydrogeno- activity. trophic methanogens is scarce. Dybas and Konisky [27] From the results obtained so far, we suggest that the F420H2 showed that methanogenesis from methyl-S-CoM and H2 is dehydrogenase transfers reducing equivalents from F420H2 to coupled to the translocation of sodium ions across the cyto- protein-bound FAD (Fig. 3). The cofactor mediated a 2e3/ plasmic membrane of Methanococcus (Mc.) voltae, indicating 1e3 switch and channels electrons to FeS clusters. The re- that ATP synthesis is mediated by a chemiosmotic mecha- maining protons are released to the periplasm by an unknown nism. The authors proposed that a membrane-associated mechanism. Reduced FeS clusters are reoxidized and the elec- H2-dependent heterodisul¢de reductase functions as a primary trons are transferred to the site of methanophenazine reduc- sodium ion pump. Such an H2 :CoB-S-S-CoM oxidoreductase tion which is envisaged to be located in the lipophilic environ- complex has been isolated from Methanobacterium (Mb.) ment of the cytoplasmic membrane. The localization of the thermoautotrophicum which contains heterodisul¢de reductase, active center would require the existence of a proton conduct- F420-non-reducing hydrogenase and several other proteins ing pathway within the protein for H‡ movement from with unknown functions [28]. However, evidence for an ion- the cytoplasm to the site of methanophenazine reduction. translocating activity has not been obtained yet. It also was The proposed mechanism would lead to the generation of shown that washed membranes of H2+CO2- or formate- a vWH‡ and would explain the stoichiometry of about grown Mc. voltae cells exhibit F420H2-dependent heterodisul- ‡ 3 2H /2e . Further electron transport within the F420H2 : ¢de reductase activity [29]. The F420-reducing hydrogenase heterodisul¢de oxidoreductase is performed by the proton- and the heterodisul¢de reductase participate in this electron translocating heterodisul¢de reductase as described above. transport system. Whether the electron transfer from F420H2 to the heterodisul¢de is coupled to energy conservation has 4.3. Formyl-methanofuran dehydrogenase system not been studied yet. The endergonic formyl-MF formation from CO2 and H2 The major di¡erence between methanogens of the order (vG0P = +16 kJ/mol) involves a membrane-bound hydro- Methanosarcinales and members of the orders Methanobac- genase, the formyl-MF dehydrogenase, and probably several teriales, Methanococcales and Methanomicrobiales is that the electron carriers. The driving force of the reaction is the trans- latter organisms do not contain cytochromes [30]. It is, there- membrane electrochemical ion potential. It is not clear yet fore, evident that redox-driven energy conservation has to be whether H‡ or Na‡ function as coupling ions [3]. di¡erent. The key enzymes of the electron transport systems The MF dehydrogenase from Ms. barkeri is composed of described in Sections 4.1 and 4.2 are also present in obligate ¢ve distinct subunits and contains molybdopterin guanine di- hydrogenotrophic methanogens. However, all of them di¡er nucleotide and several FeS clusters [2]. The genes encoding the in structure and composition from their counterparts found in subunits are organized in one operon (fmdEFACDB). Subunit methylotrophic methanogens. FmdB harbors the molybdenum-containing active site and F420-non-reducing hydrogenases from obligate hydrogeno- probably one Fe4S4 cluster. FmdF is predicted to be a poly- trophic methanogens are very similar with respect to protein ferredoxin containing eight Fe4S4 clusters and is tightly bound composition and genetic organization (for review see [12]). to the enzyme complex, indicating the important function of The enzyme from Mb. thermoautotrophicum has been ana- this polypeptide in the reaction cycle [26]. As described in lyzed in detail and can serve as a model [31]. The protein is Section 4.1, Ms. mazei Go«1 contains two operons encoding encoded by the mvhDGAB operon. The small subunit (MvhG) F420-non-reducing hydrogenases which share high homology. and the large subunit (MvhA) of the core enzyme are homol- It has been proposed that the vht-encoded hydrogenase from ogous to the corresponding polypeptides in methylotrophic

FEBS 22469 30-8-99 296 U. Deppenmeier et al./FEBS Letters 457 (1999) 291^297 methanogens (see Section 4.1). However, a signal sequence methylene-H4MPT and methyl-S-CoM [37]. Later experi- found in the gene encoding the small subunit of the F420- ments identi¢ed the methyl-H4MPT:HS-CoM methyltransfer- non-reducing hydrogenase from Ms. mazei Go«1 is not present ase as the sodium-translocating, membrane-bound, corrinoid- in mvhG. The function of MvhD, which co-puri¢es with the containing protein (Fig. 4) that catalyzes the methyl group 5 F420-non-reducing hydrogenase, is unknown. A polyferredox- transfer from N -H4MPT to HS-CoM [38,39]. Further inves- in containing eight Fe4S4 clusters is encoded by mvhB. The tigations focused on the methyl-H4MPT:HS-CoM methyl- function of this protein is still a matter of debate. It has been transferase of two organisms: Mb. thermoautotrophicum strain suggested that the protein acts as electron acceptor of the Marburg, a hydrogenotrophic methanogen, and the methylo- hydrogenase, a hypothesis which indicates that it may replace trophic Ms. mazei Go«1 (sequence data are also available from the b-type cytochromes found in the corresponding enzymes Mc. jannaschii [32] and Methanopyrus kandleri [40]). of the Methanosarcinales. However, there is still no direct Thauer and coworkers detected eight di¡erent subunits in evidence that the polyferredoxin functions as an electron car- the puri¢ed methyl-H4MPT:HS-CoM methyltransferase of rier in the H2 :CoB-S-S-CoM oxidoreductase system [9]. Mb. thermoautotrophicum; their apparent molecular masses The heterodisul¢de reductase from Mb. thermoautotrophi- are 34 (MtrH), 28 (MtrE), 24 (MtrC), 23 (MtrA), 21 cum is an iron-sulfur protein composed of the subunits HdrA (MtrD), 13 (MtrG), 12.5 (MtrB), and 12 kDa (MtrF) (80 kDa), HdrB (36 kDa) and HdrC (21 kDa) [9]. In contrast [39,41]. The mtr genes are organized in a 4.9-kbp operon in to the enzyme from Methanosarcina strains, the protein of the the order of mtrEDCBAFGH. The native enzyme forms a hydrogenotrophic organism contains FAD which is bound to tetrameric complex of 670 kDa containing 7.6 mol 5-hydrox- HdrA. Binding motifs for FeS clusters were found in HdrC ybenzimidazolyl cobamide, 37 mol non-heme iron, and 34 mol and HdrA. Parts of HdrB and HdrC are homologous to acid-labile sulfur [39]. The corrinoid prosthetic group is bound HdrD from Ms. barkeri and probably form the active site to the 23-kDa subunit [39]. The dependence of enzyme activity of the enzyme from Mb. thermoautotrophicum [9]. It was pro- on sodium ions was demonstrated [42], but the energy-con- posed that the mechanism of S-S-bond cleavage of CoM-S-S- serving nature of the protein could not be studied because a CoB is similar in the heterodisul¢de reductases from hydro- method to generate vesicular proteasomes from Mb. ther- genotrophic methanogens and from methylotrophic methano- moautotrophicum cells is not yet available. For the puri¢ed gens which was described in Section 4.1. In contrast, the methyl-H4MPT:HS-CoM methyltransferase from Ms. mazei pathway of electron transfer to the active site has to be di¡er- Go«1 we reported that the enzyme is composed of at least ent. six di¡erent polypeptides [43] containing also a cobamide 2‡=1‡ A distinct F420H2 dehydrogenase as found in Methanosar- and a [4Fe-4S] iron-sulfur cluster with an E0P value of cina strains was not detectable in Mc. voltae [29]. Further- ^426 mV [44]. The puri¢ed active enzyme was reconstituted in more, there is no indication for such an enzyme from the ether lipid liposomes of Ms. mazei Go«1 and shown to trans- analysis of the genomes from Mc. jannaschii and Mb. ther- locate sodium ions across the membrane coupled to the meth- moautotrophicum [32,33]. Other enzymes are probably in- yl transfer reaction. Stoichiometry studies have shown that ‡ volved in channeling reducing equivalents from F420H2 to approximately 2 Na are translocated per methyl group trans- membrane-integral electron carriers in hydrogenotrophic ferred [43]. Genetic analysis of the methyl-H4MPT:HS-CoM methanogens. Suitable candidates for this function are F420- methyltransferase from Ms. mazei Go«1 revealed likewise eight reducing hydrogenases. In Mc. voltae two operons were iden- coding genes organized in the same order as the operon of ti¢ed which encode F420-reducing hydrogenases of the NiFe Mb. thermoautotrophicum [45]. type (frcADGB) and the NiFeSe type (fruADGB), respectively Inhibition of the methyl transfer reaction and the concom- [34]. The puri¢ed Fru isoenzyme is composed of three sub- itant vectorial Na‡ translocation by propylation of the central units (43, 37 and 27 kDa) and contains FAD and FeS clusters. cobalt atom of the cobamide [38] gave ¢rst evidence for the Immunogold labeling revealed that the enzyme is predomi- importance of the corrinoid prosthetic group in the mecha- nantly located in the cytoplasmic membrane [35]. Recently, nism of methyl transfer. Since further experiments demon- this enzyme has been isolated from the membrane fraction strated that the methyl-H4MPT:HS-CoM methyltransferase of Mc. voltae and it was shown that the protein is involved is only active when the corrinoid exists in the cob(I) form, it in the F420H2-dependent heterodisul¢de reduction in this or- is assumed that analogous to the methionine synthase of Es- ganism [29]. cherichia coli [46] the methyl transfer proceeds in a two-step Despite the fact that the major pathways of metabolism in reaction: methylotrophic methanogens and hydrogenotrophic methano- gens are very similar, the phylogenetic di¡erences between a† CH3-H4MPT ‡ E : Cob I†amide ! these groups are re£ected in the composition of their electron transport systems. E:CH3-Cob III†amide ‡ H4MPT

6. The methyl-H4MPT:HS-CoM methyltransferase ^ b† HS-CoM ‡ E:CH3-Cob III†amide ! generation of an electrochemical sodium gradient CH3-S-CoM ‡ E : Cob I†amide First interest in sodium ions participating in the pathway of methanogenesis arose when it was discovered that growth of In the ¢rst step an enzyme-bound cob(I)amide serves as an all methanogenic archaea tested so far and formation of meth- attacking nucleophile that displaces the methyl group from ane are dependent on the presence of sodium ions [36,37]. CH3-H4MPT, resulting in a methylated enzyme and H4MPT. Early investigations using various combinations of substrates The second step corresponds to the demethylation of the en- narrowed the sodium dependence to the reactions between zyme-bound corrinoid and the methylation of HS-CoM.

FEBS 22469 30-8-99 U. Deppenmeier et al./FEBS Letters 457 (1999) 291^297 297

Although both reaction steps are accompanied by a free en- [13] Brodersen, J., Ba«umer, S., Abken, H.J., Gottschalk, G. and Dep- ergy change of 315 kJ/mol only the second step is apparently penmeier, U. (1999) Eur. J. Biochem. 259, 218^224. [14] Simianu, M., Murakami, E., Brewer, J.M. and Ragsdale, S.W. sodium ion-dependent [42]. Sodium translocation experiments (1998) Biochemistry 37, 10027^10039. with the methyl-H4MPT:HS-CoM methyltransferase con- [15] Ide, T., Ba«umer, S. and Deppenmeier, U. (1999) J. Bacteriol. 181, ¢rmed that only in the presence of HS-CoM a translocation 4076^4080. of sodium ions across the membrane occurs [43]. Various in- [16] Blaut, M., Mu«ller, V. and Gottschalk, G. (1987) FEBS Lett. 215, 53^57. vestigations demonstrated that the coordination of the cor- [17] Albracht, S.P.J. (1994) Biochim. Biophys. Acta 1188, 167^204. rinoid base to the central cobalt atom a¡ects the reactivity [18] Volbeda, A., Charon, M.H., Piras, C., Hatchikian, E.C., Frey, of the cobamide. For the methyl-H4MPT:HS-CoM methyl- M. and Fontecilla-Camps, J.C. (1995) Nature 373, 580^587. transferase of Mb. thermoautotrophicum it was demonstrated [19] Staples, C.R., Gaymard, E., Stritt-Etter, A.L., Telser, J., Ho¡- that the corrinoid is bound in the base-o¡ form combined man, B.M., Schu«rmann, P., Kna¡, D.B. and Johnson, M.K. (1998) Biochemistry 37, 4612^4620. with a histidine ligation to the protein [47]. Recent results [20] Mu«ller, V., Ruppert, C. and Lemker, T. (1999) J. Bioenerg. Bio- revealed that histidine in position 84 is the active-site histidine membr. 31, 15^27. in the MtrA subunit of the Mb. thermoautotrophicum protein [21] Haase, P., Deppenmeier, U., Blaut, M. and Gottschalk, G. [48]. EPR spectra of the Ms. mazei Go«1 methyltransferase (1992) Eur. J. Biochem. 203, 527^531. [22] Kunow, K., Linder, D., Stetter, K.O. and Thauer, R.K. (1994) yielded signal characteristics for a base-on cobamide [44]. Eur. J. Biochem. 223, 503^511. However, it was stressed that the lower ligand could be the [23] Ba«umer, S., Murakami, E., Brodersen, J., Gottschalk, G., Rags- benzimidazole base or a protein-derived ligand such as a dale, S.W. and Deppenmeier, U. (1998) FEBS Lett. 428, 295^298. histidine. In light of sequence homology it can be concluded [24] Friedrich, T. (1998) Biochim. Biophys. Acta 1364, 134^146. that cobamide binding in MtrA of Ms. mazei Go«1 also pro- [25] Majander, A., Finel, M. and Wikstro«m, M. (1994) J. Biol. Chem. 269, 21037^21042. ceeds `base o¡/his on'. [26] Vorholt, J.A. and Thauer, R.K. (1996) Eur. J. Biochem. 248, Recent studies on the methyl-H4MPT:HS-CoM methyl- 919^924. transferase are concerned with the function of single subunits [27] Dybas, M. and Konisky, J. (1992) J. Bacteriol. 174, 5575^ of the enzyme (Fig. 4). As reported above the 23-kDa subunit, 5583. [28] Setzke, E., Hedderich, R., Heiden, S. and Thauer, R.K. (1994) MtrA, harbors the essential corrinoid prosthetic group which Eur. J. Biochem. 220, 139^148. is methylated and demethylated in the catalytic cycle. The 34- [29] Brodersen, J., Gottschalk, G. and Deppenmeier, U. (1999) Arch. kDa subunit MtrH was recently demonstrated to catalyze the Microbiol. 171, 115^121. [30] Ku«hn, W., Fiebig, K., Hippe, H., Mah, R.A., Huser, B.A. and transfer of the methyl group from CH3-H4MPT to the cor- rinoid of MtrA [49]. Further on, sequence comparisons of the Gottschalk, G. (1983) FEMS Microbiol. Lett. 20, 407^410. [31] Reeve, J.N., Beckler, G.S., Cram, D.S., Hamilton, P.T., Brown, integral membrane subunit MtrD (21 kDa) indicate the J.W., Krzycki, J.A., Kolodziej, A.F., Alex, L., Orme-Johnson, presence of conserved structural elements suggesting a partic- W.H. and Walsh, C.T. (1989) Proc. Natl. Acad. Sci. 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